Human infections caused by Gram-positive bacteria present a serious therapeutic challenge due to the appearance of antibiotic-resistant strains. Of particular concern is Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis, Gram-positive organisms that are the most common cause of bacterial infections in American hospitals. These nosocomial pathogens have developed resistance mechanisms to all known antibiotic regimens and the development of novel targets for antimicrobial therapy is urgently needed. Surface proteins of Gram-positive organisms fulfill many important functions during the pathogenesis of human infections. This proposal describes the mechanism for surface protein anchoring in Gram-positive bacteria, which may serve as a target for antibacterial therapy. Staphylococcal surface proteins harbor a C-terminal sorting signal that functions first to retain polypeptides within the secretory pathway. Retention is followed by cleavage of the sorting signal between the threonine (T) and the glycine (G) of the LPXTG motif. The carboxyl of threonine is subsequently amide linked to the free amino group of peptidoglycan crossbridges, thereby anchoring the C-terminal end of surface proteins to the staphylococcal cell wall. Sortase, a membrane anchored enzyme of S. aureus, catalyzes a transpeptidation reaction, capturing cleaved surface protein as a thioester intermediate at the active site sulfhydryl. Nucleophilic attack of the amino group of pentaglycine crossbridges resolves the thioester intermediate, resulting in cell wall anchored surface protein and in regeneration of enzyme sulfhydryl. The elements and enzymes of surface protein anchoring, i.e., the LPXTG motif, the amino groups of peptidoglycan as well as sortase, are conserved in Gram-positive bacteria. This, we propose that surface protein anchoring is a universal mechanism. To test this hypothesis, we will characterize sortase function in S. aureus, E. faecalis and L. monocytogenes. Further, we propose identification of the peptidoglycan substrate of the sortase reaction, using in vivo labeling techniques as well as biochemical characterization of sorting intermediates in S. aureus, E. faecalis and L. monocytogenes. A genetic screen for S. aureus mutants defective in the retention step of surface protein anchoring will identify missing components of the cell wall sorting machinery.